All aerobic organisms use molecular oxygen for respiration and oxidation of nutrients for energy. During the reduction of molecular oxygen to water, reactive oxygen species, also know as free radicals, are generated. They are extremely reactive and can attack and damage almost all cellular components including DNA, proteins and lipids. Oxidative to stress induced by these reactive oxygen species has been implicated in many diverse degenerative diseases, including aging, arthritis, arteriosclerosis, AIDS, cancer, cataracts, hepatitis, myocardial infarction, Alzheimer's disease and stroke. To combat oxidative stress, cells have developed an arsenal of defense mechanisms including enzymes and antioxidants such as ascorbate, α-tocopherol, uric acid, β-carotene and glutathione. As the major cellular antioxidant, glutathione's metabolism and modulation is vital in the protection against oxidative stress.
Glutathione (GSH) is a tripeptide of γ-glutamic acid, cysteine and glycine, and is the most biologically abundant low-molecular weight intracellular thiol, and by way of the reducing power of its free sulphydryl (—SH), it plays a key role in many cellular processes. These include protection of cells against oxidative stress, xenobiotics and radiation. GSH is synthesized intracellularly from its constituent amino acids by the consecutive action of γ-glutamylcysteine synthetase (γ-GCS) and GSH synthetase (GS) with both reactions consuming ATP:

Lowered cellular and tissue levels of GSH is being implicated in the pathogenesis of an ever growing list of diseases and degenerative conditions, especially those associated with free radical injury. Restoration of cellular GSH levels in the treatment of a number of these medical conditions has proven to be beneficial.
Administration of GSH is considered ineffective in increasing cellular GSH as it is not effectively transported into cells and is degraded to its amino acids extracellularly. Administration of cysteine, the limiting amino acid in GSH synthesis, is also considered ineffective as it rapidly oxidizes to poorly soluble cystine and is reportedly toxic. Strategies for increasing GSH levels have focused mainly on cysteine delivery compounds such as N-acetylcysteine and 2-oxothiazalidine-4-carboxylate. Elevating GSH levels by using cysteine prodrugs is of limited use due to the feedback inhibition by GSH on the first synthetic enzyme γ-GCS. The second enzyme, GS, is not subject to feedback inhibition and its substrate, GGC, is usually the limiting substrate in GSH synthesis. γ-Glutamylcysteine (GGC) is the most immediate precursor to GSH, and GGC is normally present at lower levels than cysteine. Many cell types have the ability to transport exogenous GGC across the cell membrane and GGC has been shown to have clinical potential to elevate cellular GSH levels.
γ-Glutamylcysteine occurs naturally in bovine milk and is especially rich in the whey fraction where it occurs linked via a disulphide bond to proteins. Numerous reports have demonstrated that the consumption of whey proteins increases levels of GSH. A range of whey protein isolates is commercially available and these are claimed to include GGC as the active constituent. Recent human clinical trials on muscle performance and HIV patients have confirmed the effectiveness of whey protein isolates to increase GSH levels.
Apart from the nutraceutical market for GGC, which has already been well established by whey protein isolates, other applications for GGC include the addition of GGC to foods and cosmetics as an antioxidant, and the treatment of patients that have been poisoned with heavy metals or oxidizing agents.
To date, there is no industrially suitable, cost-effective process for the production of GGC. This is due to the complex protection and de-protection chemistry required for the reactive sulphydryl of cysteine and the unconventional γ-glutamyl peptide bond.
The majority of the processes described by the scientific and patent literature on the commercial manufacture of glutathione involve the fermentation of yeast, in particular bakers yeast, Saccharomyces cerevisiae, which is naturally rich in GSH. However, microbial production of GGC has been found to suffer from a number of disadvantages, including: low volumetric yield; low yields on substrate; extended fermentation time (several days); complex purification procedure; generation of multiple waste streams; and high equipment capital costs.
Large-scale chemical synthesis of GSH also suffers from difficulties, due to the presence of eight functional groups on the constituent amino acids. In the case of GGC there are six functional groups. Most methods for the chemical synthesis of GSH are low yielding (<10%). The major drawback preventing the commercial success of chemical synthesis of GGC is the number of steps involved: two protection steps each for both cysteine and glutamic acid, one coupling step, and then at least another two de-protection steps. Another difficulty in the chemical synthesis of GGC is that selective protection of the α-carboxyl group of glutamic acid by esterification is complicated due to the higher reactivity of the γ-carboxyl group. The inability to use platinum catalysed hydrogenolysis (de-protection) of benzyloxycarbonyl protecting groups of the amino group of glutamate in GGC, due to the poisoning effect of the sulphur from cysteine on the platinum catalyst, is also a major disadvantage. Some minor drawbacks to chemical to synthesis of GGC include the possibility of racemization of any of the two chiral centers of GGC, which is a common problem in peptide synthesis, and the environmental safety concerns inherent in using the large volumes of toxic reagents and solvents required.
Thus, there is a need for an effective process for industrial-scale production of γ-glutamylcysteine, which does not suffer from at least one or more of the above identified disadvantages, namely low yield, costly recovery, complicated and/or expensive steps, creation of multiple waste streams; lengthy fermentation steps; and high equipment capital costs.
An object of this invention is to provide a process for producing γ-glutamylcysteine or a derivative thereof in a cost effective manner by reducing the number of reaction steps, amount of raw materials used, and the loss of starting material by the formation of by-products.